Existence of a proton transfer chain in bacteriorhodopsin: Participation of Glu-194 in the release of protons to the extracellular surface

Glu-194 near the extracellular surface of bacteriorhodopsin is indispensable for proton release to the medium upon protanation of Asp-SS during light-driven transport. As for Glu-204, its replacement with glutamine (but not aspartate) abolishes both proton release and the anomalous titration of Asp-85 that originates from coupling between the pK(a) of this buried aspartate and those of the other acidic groups. Unlike the case of Glu-204, however, replacement. of Glu-194 with aspartate raises the pK, for proton release. In Fourier transform infrared spectra of the E194D mutant a prominent positive band is observed at 1720 cm(-1). It can be assigned from [4-(13)C]aspartate and D(2)O isotope shifts to the C=O stretch of protonated Asp-194. Its rise correlates with proton transfer from the retinal Schiff base to Asp-85. Its decay coincides with the appearance of a proton at the surface, detected under similar conditions with fluorescein covalently bound to Lys-129 and with pyranine. Its amplitude decreases with increasing pH, with a pK(a) of about 9. We show that this pK(a) is likely to be that of the internal proton donor to Asp-194, the Glu-204 site, before photoexcitation: while (13)C NMR titration indicates that Asp-194 has an initial pK(a) of about 3. We propose that there is a chain of interacting residues between the retinal Schiff base and the extracellular surface. After photoisomerization of the retinal the pK(a)'s change so as to allow (i) Asp-85 to become protonated by the Schiff base, (ii) the Glu-204 site to transfer its proton to Asp-194 in E194D, and therefore to Glu-194 in the wild type, and (iii) residue 194 to release the proton to the medium.